Despite major advances in medicine in general, and in the field of oncology in particular, cancer remains a leading cause of death worldwide. In 2007, it accounted for 7.9 million fatalities or 13 of global mortality. Amongst women, breast cancer has the highest incidence, causing 548,000 deaths per annum. Hence, the ‘war on cancer’, declared by US president Nixon some four decades ago, is far from over.
A tumor is an abnormal growing cell mass, resulting from either an increased input of cells or a decreased output. Nonetheless, primary tumor growth is no longer the major therapeutic problem. Particularly for ‘benign’ or ‘non-aggressive’ tumors, surgical removal, radiotherapy, chemotherapy and immunotherapy have proven to be successful treatments for a great number of patients.
Fundamentally different, however, are ‘malignant’ or ‘fast progressive’ cancers, which are capable of invading into neighboring tissues, and subsequently spread to locoregional or distant organs, causing secondary tumors in permissive tissues. These processes, invasion and metastasis, are the foremost causes of cancer deaths and accordingly constitute a main challenge in cancer research. The molecular mechanisms of tumor invasion have been partly unraveled and originate from a disequilibrium between the expression of invasion promoter and invasion suppressor genes (Mareel et al., 1994). Interestingly, although invasion is crucial at all the different steps of the metastatic process, not every invasive tumor has a tendency to metastasize.
Existing studies or therapies are focusing on inhibition of mitosis (via microtubule assembly) and on proliferation (Edwards et al., 1990; Romagnoli et al. 2008; Bath et al., 2005; WO02/02593), or on inhibition of angiogenesis, the recruitment of new blood vessels (Robinson et al., 2005; WO02/02593; WO03/037315). In striking contrast to the new, efficient generation of growth-inhibiting drugs targeting specific growth signaling pathways in tumor cells (e.g. trastuzumab, imatinib and farnesyl transferase inhibitors), there are still no similar pharmaceuticals available to treat or prevent invasion and metastasis. Many anti-neoplastic compounds are designed to disrupt replication in rapidly dividing cells, or to inhibit a key metabolic link in actively proliferating cells. Although such approaches have met with levels of success in certain types of cancers, or cancers at certain stages, chemotherapy is generally associated with unpleasant to debilitating side effects due to their effects on normal cells, such as malaise, nausea, loss of appetite, alopecia, and anemia, and in the extreme, loss of immune function and/or loss of digestive activity. Further, compounds which act at the level of cell replication, either by introducing nucleotide analogs into dividing cells, or by disrupting normal replication, have the potential of introducing widespread genetic mutations in normal cells in the subject. In addition, cancer cells may develop resistance to many types of anti-cancer agents, either by limiting uptake of the agent into the cells, or by altering the metabolism of the agent within the cells.
Although angiogenesis inhibitors are designed to prevent the formation of new blood vessels, thereby stopping or slowing down the growth or spread of tumors, use of said compounds will not always result in the inhibition of invasion, since invasion can occur without angiogenesis. Even more, Pennaccietti et al. state that although angiogenesis inhibitors have shown promise in hindering blood supply and holding tumors in check it seems that such inhibitors, by depriving tumors of oxygen, could have an unintended effect, i.e. promotion of metastasis. Hence tumor cell resistance to antiangiogenic therapy through sustained viability or increased invasion may represent a previously unrecognized challenge.
At present, bisphosphonates are the most frequently applied drugs to treat bone metastases. Besides, microtubule (Atassi et al., 1982) and angiogenesis inhibitors (Jain et al., 2006) have been included in clinical trials to evaluate their anti-invasive and anti-metastatic activity.
Nevertheless, there is a continuous need for compounds specifically targeting the first steps of malignant tumor development, in particular inhibiting invasion. By specifically targeting the ability of tumor cells to invade neighboring tissue, an otherwise malignant tumor may be arrested at its initial site, and prevented from spreading throughout the body.
It was therefore an aspect of the present invention to provide compounds having specific anti-invasive activity. Furthermore, as for most pharmaceutical compounds, it is desirable to provide compounds having a lowest active concentration which is as low as possible to avoid undesired side-effects but still obtain the desired activity. In general in order to determine the lowest active concentration of a compound each individual compound needs to be synthesized and tested in vitro at different concentrations. However, as evident said method is far from efficient for testing a large set of compounds to select those having the best chance of success. It was therefore a further objective of this invention to provide a method suitable for in silico determining the lowest active concentration of a compound, thereby significantly reducing the number of potentially interesting compounds to be synthesized and tested in vitro.
In our quest for new anti-invasive agents, we have recently adopted a dual strategy of synthesis and modeling. Firstly, in search of new leads, we analyzed the results of the screening program of the Bracke group (Ghent University Hospital, Belgium), containing in vitro anti-invasive activity data for hundreds of compounds. Although few of the screened substances inhibit invasion of healthy tissues by MCF-7/6 carcinoma cells, the potentially interesting agents belong to very diverse categories: polyphenolics, peptides, steroids, phospholipids and retinoids. The promising potential of the polyphenolics was reflected in the establishment of the Indo-Belgian screening program, a collaborative effort of the Universities of Delhi and Ghent, which has systematically assessed the in vitro anti-invasive activity of polyphenolics and alkaloids since 1989 (Vanhoecke et al., 2005). Looking closer at the results of this collaboration, our attention was primarily drawn towards chalcones (1,3-diarylpropenones) and structurally related compounds, as most of these were shown to have an anti-invasive activity at 1 or 10 μM.
Although certainly promising as a set of lead compounds, no systematic substitution pattern was available in this initial library, since the tested chalcones were all natural products. Therefore, our first aim was to broaden the series of screened chalcones by synthesizing (unnatural) analogues. However, since the binding site(s) of the chalcones remain(s) unrevealed, the design of new compounds based on the topology of the receptor was impossible. As the preparation and biological evaluation of a complete library of chalcones, comprising a wide range of substituents and pharmacophores, would be an enormous task, we envisaged synthesizing a limited number of compounds possessing a strategically chosen substitution pattern. The broadened library could then be used to develop a quantitative structure-activity relationship (QSAR) model, which predicts the anti-invasive activity of hypothetical compounds. On its turn, this in silico screening would enable us to focus future synthetic efforts on promising substances. Such use of QSAR approaches is attractive from an economic as well as an ecological point of view.
As further detailed in the examples that follow herein after, this QSAR approach allowed us to provide a model for the in silico prediction of the anti-invasive activity of chalcone-like compounds. It further allowed us to identify potentially interesting anti-invasive chalcone-like compounds having a predicted anti-invasive activity at a lowest active concentration of ≦0.1 μM and thus being at least 10-100 times more effective in comparison to chalcone-like compounds which are currently under investigation for their use as anti-invasive compounds.
Katritzky et al., 2006 also describes a QSAR model for determining the anti-invasive activity of multiple organic compounds including chalcone-like compounds. However, said model is not suitable for predicting anti-invasive compounds having a lowest active concentration of less than 1 μM, since the lowest active concentration which can be predicted making use of this model is set at 1 μM, as evident from table 1 of this publication and from Bracke et al., 2008.
Yadav et al., 2011 (table 4) provides an overview of multiple chalcone-like compounds and their lowest active concentration rendering “anti-cancer” activity, said concentrations ranging from 1.9 to 50 μM. These compounds are thus at least 20 times less effective compared to the compounds according to this invention.
Furthermore, also Abel Bar et al., 2010 (lowest active concentration 1 μM), Mukherjee et al., 2010 (lowest active concentration 10 μM), Parmar et al., 1997 (lowest active concentration 1 μM), Parmar et al., 1998 (lowest active concentration 1 μM) and Parmar et al., 2003 (lowest active concentration 1 μM) provide chalcone-like compounds which were tested for their anti-invasive activity, however as evident from these documents, none of said compounds have been shown to have anti-invasive activity of less than 1 μM.
In summary, it is generally known that the higher the concentration of a compound needed to obtain a certain effect, the higher the risk for undesired side-effects. Therefore, there was a need for compounds having an anti-invasive activity at the lowest possible concentration, as well as for a model to predict said anti-invasive activity in order to reduce the burden of experimentation. The method and compounds according to this invention provide a solution to said problems. More in particular, the present invention provides chalcone-like compounds having an anti-invasive activity at a concentration of 0.1 μM, or even less. Moreover, these compounds are not toxic and have no negative effect on the survival of the cells. It is a further advantage that these compounds have a broad field of application, i.e. they can be used for the treatment of several types of cancers, more specific cancers characterized by the presence of a malignant solid tumor.